The invention relates to methods for regulating cell motility and related products. In particular methods for promoting and preventing cell migration are described herein.
How a cell moves is one of the most compelling mysteries of cell biology. Cell migration forms the basis for higher order processes such as immune cell homing, wound healing, and axonal pathfinding. Migration depends on the coordinated execution and integration of complex individual processes. Although different cell types have unique approaches to cell movement, it is useful to consider animal cell migration in a generalized way. In its simplest form, movement requires that a cell generates and maintains a state of asymmetry or polarity.
Once polarized, a cell must execute a four-step cycle to migrate or translocate (reviewed in Lauffenburger, D. A., and Horwitz, A. F. (1996). Cell migration: a physically integrated molecular process. Cell 84, 359-69). First, a cell must extend a process, known as the leading edge, in the direction of movement. During this step, increased actin polymerization is seen in the area of the leading edge. This increased polymerization arises from the creation of new barbed ends that are oriented towards the membrane, either by nucleation of new filaments from pools of G-actin or by severing or uncapping of existing filaments. Actin monomers are added onto barbed ends until they are capped (Schafer, D. A., and Cooper, J. A. (1995). Control of actin assembly at filament ends. Annu Rev Cell Dev Biol 11, 497-518). The combination of actin nucleation and filament elongation is thought to play a critical role in the protrusion of the leading edge (Eddy, R. J., Han, J., and Condeelis, J. S. (1997). Capping protein terminates but does not initiate chemoattractantxe2x80x94induced actin assembly in Dictyostelium. J Cell Biol 139, 1243-53). Second, once a cell has extended a process, it must form semi-stable points of attachment with the underlying substratum to serve as anchor points. One class of attachment points, focal adhesions, contain aggregates of integrin receptors and a variety of cytosolic signaling and cytoskeletal proteins and serve as sites of bidirectional signaling between the extracellular matrix and the actin cytoskeleton (Schoenwaelder, S. M., and Burridge, K. (1999). Bidirectional signaling between the cytoskeleton and integrins. Curr Opin Cell Biol 11, 274-86). Although attachment of newly extended processes may be critical for cell translocation, process extension itself does not require adhesion (Bailly, M., Yan, L., Whitesides, G. M., Condeelis, J. S., and Segall, J. E. (1998). Regulation of protrusion shape and adhesion to the substratum during chemotactic responses of mammalian carcinoma cells. Exp Cell Res 241, 285-99). Third, once a cell has extended and anchored a new process, it must slide the cell body forward by traction. The fourth step is release of points of substratum attachment at the rear of the cell.
The evolutionarily-conserved Ena/VASP protein family has been implicated in the regulation of cell migration (Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J., and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87, 227-39). Enabled (Ena; SEQ ID NO: 9) was identified as a genetic suppressor of loss-of-function mutations in Drosophila Ableson tyrosine kinase (D-Ab1) (Gertler, F. B., Doctor, J. S., and Hoffinann, F. M. (1990). Genetic suppression of mutations in the Drosophila abl proto-oncogene homolog. Science 248, 857-60). Loss-of-function mutations in Ena ameliorated the embryonic central nervous system defects associated with loss of D-Ab1 in combination with mutations in any of several known D-Ab1 modifier genes (Gertler, F. B., Corner, A. R., Juang, J L., Ahern, S. M., Clark, M. J., Liebl, E. C., and Hoffmann, F. M. (1995). enabled, a dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine kinase, encodes an Abl substrate with SH3 domain-binding properties. Genes Dev 9, 521-33). VASP was identified biochemically as an abundant substrate for cyclic-nucleotide dependent kinases in mammalian platelets (SEQ ID NO: 10); (Halbrugge, M., and Walter, U. (1990). Analysis, purification and properties of a 50,000-dalton membrane- associated phosphoprotein from human platelets. J Chromatogr 521, 335-43). Two other mammalian members of this protein family, Mena (mammalian Enabled; SEQ ID NO: 2 and EVL (Ena/VASP like; SEQ ID NO: 11), were identified by sequence similarity (Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J., and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87, 227-39).
All Ena/VASP family members share a conserved domain structure. The N-terminal third of the protein, the EVH1 (Ena VASP Homology) domain (Gertler, F. B., Niebuhr, K., Reinhard, M, Wehland, J, and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87, 227-39), mediates subcellular targeting of Ena/VASP proteins to focal adhesions by binding to proteins containing a motif whose consensus is D/E FPPPPX D/E (SEQ ID NO: 1) (Niebuhr, K., Ebel, F., Frank, R., Reinhard, M., Domann, E., Carl, U. D., Walter, U., Gertler, F. B., Wehland, J., and Chakraborty, T. (1997). A novelproline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligandfor the EVH1 domain, a protein module present in the Ena/VASP family. Embo J 16, 5433-44). Mutational analysis indicated that the phenylalanine residue, along with flanking acidic residues on either side, are critical for optimal binding (Carl, U. D., Pollmann, M., Orr, E., Gertler, F. B., Chakraborty, T., and Wehland, J. (1999). Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands. Curr Biol 9, 715-8). The EVH1 ligand motif is found in a number of cellular proteins, including the focal adhesion proteins zyxin and vinculin. The central portion of Ena/VASP proteins contains proline-rich stretches, which have been reported to be binding sites for three types of proteins: the G-actin binding protein profilin, SH3 domain-containing proteins, and WW domain-containing proteins (Ermekova, K. S., Zambrano, N., Linn, H., Minopoli, G., Gertler, F., Russo, T., and Sudol, M. (1997). The WW domain of neural protein FE65 interacts with proline-rich motifs in Mena, the mammalian homolog of Drosophila enabled. J Biol Chem 272, 32869-77; Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J., and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87, 227-39). The C-terminal third of Ena/VASP proteins contains the EVH2 domain that binds in vitro to F-actin and has a putative coiled-coil region reported to be important for multimerization (Bachmann, C., Fischer, L., Walter, U., and Reinhard, M. (1999). The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation. J Biol Chem 274, 23549-57,; Huttelmaier, S., Harbeck, B., Steffens, O., Messerschmidt, T., Illenberger, S., and Jockusch, B. M. (1999). Characterization of the actin binding properties of the vasodilator-stimulatedphosphoprotein VASP. FEBS Lett 451, 68-74).
In addition to their capacity to bind profilin and actin, the localization of Ena/VASP proteins suggests that they may be involved in regulating actin dynamics and/or adhesion. In fibroblasts, Ena/VASP proteins are localized to focal adhesions, in a weak punctuate pattern along stress fibers and to the leading edge, while in neuronal growth cones, they are concentrated at the distal tips of filopodia (Reinhard, M., Halbrugge, M., Scheer, U., Wiegand, C., Jockusch, B. M., and Walter, U. (1992). The 46/50 kDa phosphoprotein VASP purifiedfrom human platelets is a novel protein associated with actin filaments and focal contacts. Embo J. 11, 2063-70; Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J., and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87, 227-39; Lanier, L. M., Gates, M. A., Witke, W., Menzies, A. S., Wehman, A. M, Macklis, J. D., Kwiatkowski, D., Soriano, P., and Gertler, F. B. (1999). Mena is required for neurulation and commissure formation. Neuron 22, 313-25). Genetic analyses of Ena/VASP family members in flies and mice demonstrated that these proteins function in processes that involve cell shape change, and movement including platelet aggregation and axon guidance (Aszodi, A., Pfeifer, A., Ahmad, M., Glauner, M., Zhou, X. H., Ny, L., Andersson, K. E., Kehrel, B., Offermanns, S., and Fassler, R. (1999). The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function. Embo J. 18, 37-48; Wills, Z., Bateman, J., Korey, C. A., Corner, A., and Van Vactor, D. (1999). The tyrosine kinase Abl and its substrate enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance. Neuron 22, 301-12). In mice, a dosage-sensitive genetic interaction between Mena and profilin I supports a model in which these two proteins function in concert during development (Lanier, L. M., Gates, M. A., Witke, W., Menzies, A. S., Wehman, A. M., Macklis, J. D., Kwiatkowski, D., Soriano, P., and Gertler, F. B. (1999). Mena is required for neurulation and commissure formation. Neuron 22, 313-25).
Ena/VASP proteins are also implicated in actin dynamics by their role in facilitating the actin-based motility of the intracellular bacterial pathogen Listeria monocytogenes. The Listeria protein, ActA is required for the formation of actin tails characteristic of motile bacteria (Kocks, C., Gouin, E., Tabouret, M., Berche, P., Ohayon, H., and Cossart, P. (1992). L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein. Cell 68, 521-31; Domann, E., Wehland, J., Rohde, M., Pistor, S., Hartl, M., Goebel, W., Leimeister-Wachter, M., Wuenscher, M., and Chakraborty, T (1992). A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin. Embo J 11, 1981-90). Furthermore, the motility of the intracellular pathogen Listeria monocytogenes resulting from rapid actin polymerization at one pole of the bacterium requires Ena (Laurent, V., Loisel, T. P., Harbeck, B., Wehman, A., Grobe, L., Jockusch, B. M., Wehland, J., Gertler, F. B., and Carlier, M. F. (1999). Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. J. Cell Biol 144, 1245-58; Loisel, T. P., Boujemaa, R., Pantaloni, D., and Carlier, M. F. ( 999). Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 401, 613-6).
ActA is a multi-domain protein on the surface of the bacteria that interacts with host cell factors to trigger actin assembly (Pistor, S., Chakraborty, T., Walter, U., and Wehland, J. (1995). The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins. Curr Biol 5, 517-25). Actin nucleation is driven by ActA-mediated activation of the Arp2/3 complex (Welch, M. D., Rosenblatt, J., Skoble, J., Portnoy, D. A., and Mitchison, T. J. (1998). Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actinfilament nucleation. Science 281, 105-8). Ena/VASP proteins are the only host cell factors known to bind directly to ActA in vivo, which contains four optimized copies of the D/E FPPPPXDDE (SEQ ID NO: 1) EVH1 ligand motif (Niebuhr, K., Ebel, F., Frank, R., Reinhard, M., Domann, E., Carl, U. D., Walter, U., Gertler, F. B., Wehland, J., and Chakraborty, T. (1997). A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family. Embo J. 16, 5433-44). Mutation of these repeats leads to a defect in bacterial movement, despite the fact that an actin cloud and short actin tails still form around the bacterium (Smith, G. A., Theriot, J. A. and Portnoy, D. A., 1996. The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulatedphosphoprotein and profilin. J. Cell Bio. 135:647-660; Niebuhr, K., Ebel, F., Frank, R., Reinhard, M., Domann, E., Carl, U. D., Walter, U., Gertler, F. B., Wehland, J., and Chakraborty, T. (1997). A novelproline-rich motifpresent in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the EnalVASP family. Embo J 16, 5433-44). In vitro experiments using either depleted cell-free extracts or reconstitution with purified proteins directly demonstrated that Ena/VASP are required for efficient actin tail formation and normal bacterial motility (Laurent, V., Loisel, T. P., Harbeck, B., Wehman, A., Grobe, L., Jockusch, B. M., Wehland, J., Gertler, F. B., and Carlier, M. F. (1999). Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. J Cell Biol 144, 1245-58; Loisel, T. P., Boujemaa, R., Pantaloni, D., and Carlier, M. F. (1999). Reconstitution of actin-based motility ofListeria and Shigella using pure proteins. Nature 401, 613-6). It has been proposed that Ena/VASP proteins act to increase the rate of actin filament extension by increasing the local pool of profilin-actin complexes (Beckerle, M. C. (1998). Spatial control of actinfilament assembly: lessons from Listeria. Cell 95, 741-8). Listeria has been proposed as a model for the reorganization of actin at the leading edge of a motile cell. Recent work using GFP-tagged VASP demonstrated a strong correlation between membrane extension rates and the concentration of VASP at the leading edge (Rottner, K., Behrendt, B., Small, J. V., and Wehland, J. (1999). VASP dynamics during lamellipodia protrusion. Nat Cell Biol 1, 321-2). Based on localization studies and the Listeria experiments, it has been proposed that Ena/VASP proteins serve to promote actin-based cell movement.
U.S. Pat. No. 5,990,087 issued to Lal et al., describes a human Ena/VASPxe2x80x94like protein splice variant referred to as EVL1 and methods of use thereof. The patent teaches that EVL1 has an activity which is similar to the activity that has been proposed for the known Ena/VASP proteins. Specifically, the patent teaches that EVL1 antagonists which reduce EVL1 activity within a cell can be used to treat or prevent cancer and EVL1 agonists which increase EVL1 activity within a cell can be used to treat or prevent a nervous system disorder.
The invention relates, in some aspects, to methods for promoting or preventing cellular migration and for various therapeutic treatments using Ena/VASP inhibitors and activators. It has been discovered, surprisingly, that Ena/VASP proteins are negative regulators of cell motility. Because of the role of Ena/VASP proteins in the positive regulation of cell motility in the Listeria system and because of the localization of Ena/VASP in focal adhesions and neuronal growth cones it was widely believed in the prior art that Ena/VASP proteins are universally positive regulators of cell motility. The prior art such as U.S. Pat. No. 5,990,087 hypothesized that Ena/VASP proteins play a positive role in regulating cell motility and thus inhibition of these proteins should be beneficial for the treatment of cancer by reducing or eliminating cellular migration and thus metastasis. In contrast to the teachings of the prior art, it was discovered that Ena/VASP proteins are actually negative regulators of cell motility. When Ena/VASP protein activity is upregulated cell motility is reduced significantly. Alternatively when Ena/VASP protein activity is downregulated cell motility is enhanced significantly. Thus, it has been discovered according to the invention that upregulation of Ena/VASP protein activity can be used to slow cellular migration and thus to prevent cell metastasis and that downregulation of Ena/VASP protein activity can be used to increase cell motility to promote would healing and tissue regeneration.
In one aspect the invention is a method for preventing mammalian cell migration. The method involves inducing a functional Ena/VASP protein in a mammalian cell in an effective amount for preventing cell migration. In other aspects the invention is a method for preventing tumor cell metastasis in a subject. The method involves administering to a subject having or at risk of developing a metastatic cancer a plasma membrane targeting compound in an effective amount for preventing cell migration in order to prevent tumor cell metastasis. In yet other aspects, the invention is a method for preventing or treating inflammatory disease in a subject. The method involves administering to a subject having or at risk of developing an inflammatory disease a plasma membrane targeting compound in an effective amount for preventing cell migration in order to prevent or treat the inflammatory disease.
In some preferred embodiments the functional Ena/VASP protein is induced by contacting the mammalian cell with an Ena/VASP activator. The Ena/VASP activator can be a plasma membrane targeting compound that targets the endogenous Ena/VASP protein to the plasma membrane or in other embodiments it can be exogenous EDa/VASP protein. The plasma membrane targeting compound may be an Ena/VASP binding molecule conjugated to a plasma membrane targeting domain. Optionally the Ena/VASP binding molecule is an EVH1 binding molecule. EVH1 binding molecules include but are not limited to FPPPP peptides (SEQ ID NO.: 3) and peptide mimetics. In other embodiments the functional Ena/VASP protein is induced by expression of exogenous Ena/VASP protein in the cell.
The mammalian cell may be any type of cell but in some embodiments is a tumor cell. The tumor cell may be a tumor cell that is treated in vitro or in vivo.
The Ena/VASP protein may be any type of Ena/VASP protein known in the art, including proteins having homology to known Ena/VASP proteins. In some embodiments the Ena/VASP protein is a protein selected from the group consisting of Mena, VASP and Evl.
The invention in another aspect involves a method for promoting cell migration. The method is performed by depleting a mammalian cell of a functional Ena/VASP protein to promote cell migration. In some embodiments the functional Ena/VASP protein is depleted by contacting the mammalian cell with an Ena/VASP inhibitor.
In another related aspect the invention is a method for promoting wound healing. The method involves contacting a mammalian cell involved in wound healing with an Ena/VASP inhibitor to promote migration of the mammalian cell to the site of the wound. In some embodiments the Ena/VASP inhibitor is administered in vivo to a subject at the site of the wound.
The invention in other aspects relates to a method for promoting tissue generation. The method involves contacting mammalian cells of a tissue type with an Ena/VASP inhibitor to promote actin polymerization and tissue formation on a scaffold. In some embodiments the scaffold is an artificial scaffold in vitro and optionally the scaffold is implanted in vivo once the tissue has generated. In other embodiments the scaffold is an artificial scaffold in vivo. Optionally the scaffold is a naturally occurring tissue scaffold in vivo. In other embodiments the Ena/VASP inhibitor is administered to a site of damaged nerve cells in a subject on a naturally occurring tissue scaffold.
In some preferred embodiments the Ena/VASP inhibitor is an Ena/VASP binding molecule conjugated to an intracellular targeting domain that targets Ena/VASP protein to a surface remote from the plasma membrane. The Ena/VASP binding molecule preferably is an EVH1 binding molecule, which may optionally be a FPPPP peptide (SEQ ID NO.: 3) or a peptide mimetic. In other embodiments the Ena/VASP inhibitor is an Ena/VASP antisense molecule.
The mammalian cell may be any type of cell. In some preferred embodiments the mammalian cell is a fibroblast, a nerve cell, a glial cell, an epithelial cell, an endothelial cell and a muscle cell. In some embodiments the cell is a fibroblast and the fibroblast is contacted with the Ena/VASP inhibitor in vitro and in other embodiments the fibroblast is applied to the site of a wound in vivo.
The Ena/VASP protein may be any type of Ena/VASP protein known in the art, including proteins having homology to known EnaNASP proteins. In some embodiments the Ena/VASP protein is a protein selected from the group consisting of Mena, VASP and Evl.
According to other aspects of the invention a method for promoting tissue regeneration. In some embodiments this method is useful for preventing or treating neurodegenerative diseases. The method involves administering to a subject having or at risk of neurodegeneration an Ena/VASP inhibitor in an amount effective to promote tissue regeneration or to prevent neurodegeneration.
In some embodiments the Ena/VASP inhibitor is administered locally to the site of tissue where generation is desired or to the site of neurodegeneration. In other embodiments the Ena/VASP inhibitor is administered to a nerve cell in vitro and the nerve cell is delivered to the subject at the site of neurodegeneration. In yet other embodiments the Ena/VASP inhibitor is administered in a sustained release vehicle at the site of neurodegeneration.
Preferably the subject having or at risk of neurodegeneration has or is at risk of developing Alzheimer""s disease, Down Syndrome; Parkinson""s disease; amyotrophic lateral sclerosis (ALS), stroke, direct trauma, Huntington""s disease, epilepsy, ALS-Parkinsonism-dementia complex; progressive supranuclear palsy; progressive bulbar palsy, spinomuscular atrophy, cerebral amyloidosis, Pick""s atrophy, Retts syndrome; Wilson""s disease, Striatonigral degeneration, corticobasal ganglionic degeneration; dentatorubral atrophy, olivo-pontocerebellar atrophy, paraneoplastic cerebellar degeneration; Tourettes syndrome, hypoglycemia; hypoxia; Creutzfeldt-Jakob disease; or Korsakoff s syndrome.
In one embodiment the Ena/VASP inhibitor is administered in an effective amount for preventing EnalVASP proteins from interacting with FE65, profilinl or profilin2.
The invention also relates to methods of enhancing or disrupting learning and memory. It was discovered according to the invention that inhibition of Ena/VASP proteins is sufficient to enhance learning and memory and also that activation of Ena/VASP proteins is sufficient to disrupt learning and memory. Thus in one aspect the invention relates to a method for enhancing learning and memory by administering to a subject an Ena/VASP inhibitor in an amount effective to enhance learning and memory. In some embodiments the subject has or is at risk of developing a learning disorder selected from the group consisting of Alzheimer""s disease, Creutzfeld-Jakob disease, brain damage, senile dementia, Korsakow""s disorder, and age-related memory loss. In other embodiments the Ena/VASP inhibitor is administered in an effective amount for inhibiting the activity of Mena in a synapse of the subject. In another aspect the invention relates to a method for disrupting learning and memory by administering to a subject an Ena/VASP activator in an amount effective to disrupt learning and memory.
The inhibitor or activator can be administered systemically or locally and in some embodiments is specifically targeted to the brain.
The invention also encompasses compositions and kits. In one aspect the invention is a composition of an Ena/VASP inhibitor in an effective amount for promoting cellular migration and a pharmaceutically acceptable carrier and in other aspects the invention is a composition of an effective amount for preventing cellular migration, of an Ena/VASP activator in a pharmaceutically acceptable carrier.
According to other aspects of the invention methods for identifying a therapeutic Ena/VASP activator or inhibitor are provided. The method involves contacting a mammalian cell with a putative Ena/VASP activator or inhibitor and either determining the effect of the putative Ena/VASP activator or inhibitor on cell migration or determining the intracellular location of endogenous Ena/VASP. The Ena/VASP activator or inhibitor is identified by observing either a decreased rate of migration or an increased rate of migration with respect to an untreated control mammalian cell respectively or altered localization.
The putative Ena/VASP activator or inhibitor may be obtained from any source but in preferred embodiments it is obtained from a peptide library of compounds, a small molecule library of compounds, or a peptidomimetic library of compounds. In other embodiments the putative Ena/VASP activator or inhibitor is obtained from a mixture of compounds identified using an anti-idiotypic antibody.
The invention also includes modified cells and screening assays based on those cells. Thus in one aspect the invention is a modified cell which is an Ena/VASP double negative cell. Preferably the cell is a fibroblast and preferably the cell is a Mena/VASP double mutant. In one embodiment the cell is a mammalian cell. The cell may be used in a method for identifying a therapeutic compound for inhibiting cellular migration. The method involves contacting the modified cell with a putative compound for inhibiting cellular migration, and determining the effect of the putative compound on cellular migration, wherein a putative compound which inhibits cellular migration is a therapeutic compound.
In yet other aspects the invention relates to a compound having an actin binding domain and a cell motility domain, but which does not include a Listeria motility domain. In one embodiment the actin binding domain is a peptide sequence corresponding to amino acids 411-429 of Mena or a conservative substitution thereof. In another embodiment the cell motility domain includes a conserved EVH1 domain. In preferred embodiments the cell motility domain is a peptide sequence corresponding to amino acids 1-280 of Mena or a conservative substitution thereof. The compound may be a peptide having a sequence corresponding to a conservative substitution thereof. In yet other embodiments the compound is a mimetic.
A modified Ena/VASP protein is also provided according to aspects of the invention. The modified Ena/VASP protein includes the amino acid sequence of the mature peptide of SEQ ID NO: 2 wherein at least one amino acid residue has been substituted and wherein the substitution is selected from the group consisting of (a) a non-conservative or conservative substitution of a serine residue corresponding to position 236 or 376 of SEQ ID NO: 2; (b) a non-conservative substitution or deletion of one or more residues corresponding to position 411-429 of SEQ ID NO: 2; (c) a conservative substitution of at least one residue corresponding to position 281-344 of SEQ ID NO: 2; (d) a non-conservative substitution or deletion of at least one residue corresponding to position 281-344 of SEQ ID NO: 2; and (e) a non-conservative or conservative substitution or deletion of one or more residues corresponding to position 411-429 of SEQ ID NO: 2.
The invention in another aspect is a method for identifying a therapeutic compound for inhibiting or promoting cellular migration. The method involves screening one or more putative compounds for the ability to interact with an actin barbed end to identify an actin binding molecule, and determining the effect of the actin binding molecule on cellular migration to determine whether the actin binding molecule is a therapeutic compound for inhibiting or promoting cellular migration. In one embodiment a composition identified by the method is included in the invention.
Each of the embodiments of the invention can encompass various recitations made herein. It is, therefore, anticipated that each of the recitations of the invention involving any one element or combinations of elements can, optionally, be included in each aspect of the invention.